www.elsevier.com/locate/ghir

Growth Hormone & IGF Research 16 (2006) 29–40

Growth hormone- and pressure overload-inducedcardiac hypertrophy evoke different responses toischemia-reperfusion and mechanical stretch

Hinrik Stromer a, Emiliano A. Palmieri b, Mark C.H. De Groot a,Francesca Di Rella b, Andrea Leupold a, Michael Horn a, Maria G. Monti b,

Raffaele Napoli b, Angela Di Gianni b, Jorgen Isgaard c, Luigi Sacca b,Stefan Neubauer d, Antonio Cittadini b,*

a Department of Medicine, Medizinische Universitatsklinik Wurzburg, 97080 Wurzburg, Germanyb Department of Internal Medicine and Cardiovascular Sciences, University Federico II, Via S. Pansini, 5, 80131 Naples, Italy

c Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital, S-413 45 Goteborg, Swedend University of Oxford Centre for Clinical Magnetic Resonance Research, Department of Cardiovascular Medicine,

John Radcliffe Hospital, OX3 9DU Oxford, UK

Received 28 January 2005; revised 5 September 2005; accepted 6 September 2005Available online 3 November 2005

Abstract

Objective. To compare the molecular, histological, and functional characteristics of growth hormone (GH)- and pressure over-load-induced cardiac hypertrophy, and their responses to ischemia-reperfusion and mechanical stretch.Design. Four groups of male Wistar rats were studied: aortic banding (n = 24, AB) or sham (n = 24, controls) for 10 weeks, and GHtreatment (n = 24; 3.5 mg/kg/day, GH) or placebo (n = 24, controls) for 4 weeks. At 13 weeks, the rats were randomly subjected to:(i) assessment of basal left ventricular mRNA expression of sarcoplasmic reticulum calcium-ATPase (SERCA-2), phospholamban(PLB), and Na+–Ca2+ exchanger (NCX) and collagen volume fraction (CVF) (Protocol A, 8 rats in each group); (ii) left ventricularno-flow ischemia with simultaneous evaluation of intracellular Ca2+ handling and ATP, phosphocreatine (PCr) and inorganic phos-phate (Pi) content (Protocol B, 12 rats in each group); or (iii) left ventricular mechanical stretch for 40 min with assessment of tumornecrosis-a (TNF-a) mRNA (Protocol C, 4 rats in each group). Protocol B and C were carried out in a Langendorff apparatus.Results. In Protocol A, no difference was found as to myocardial mRNA content of Ca2+ regulating proteins and CVF in GHanimals vs controls. In contrast, in the AB group, myocardial mRNA expression of SERCA-2 and PLB was downregulated whilethat of NCX and CVF were increased vs. controls (p < 0.05). In Protocol B, recovery of left ventricular function was significantlydecreased in AB vs GH groups and controls and this was associated with 1.6-fold increase in intracellular Ca2+ overload duringreperfusion (p < 0.05). Baseline ATP content was similar in the four study groups, whereas PCr and Pi was lower in AB vs GHrats and controls. However, the time courses of high-energy phosphate metabolic changes did not differ during ischemia andreperfusion in the four study groups. In Protocol C, no detectable TNF-a mRNA level was found in the left ventricular myo-cardium of GH treated rats and controls at baseline, while a modest expression was noted in AB animals. Mechanical stretchresulted in de novo myocardial TNF-a mRNA expression in GH group and controls, which was dramatically increased inAB animals (�5-fold above baseline, p < 0.001).Conclusions. The data show that cardiac hypertrophy activated by short-term GH treatment confers cardioprotection comparedwith pressure overload with regard to molecular and histological characteristics, and responses to ischemia-reperfusion and mechan-ical stretch.� 2005 Elsevier Ltd. All rights reserved.

1096-6374/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ghir.2005.09.002

* Corresponding author. Tel.: +39 81 7464375; fax: +39 81 7463199.E-mail address: antonio.cittadini@unina.it (A. Cittadini).

30 H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40

Keywords: Ischemia-reperfusion; Calcium handling; Cytokines; Somatotropin; Hypertrophy

1. Introduction

Augmenting muscles mass is one of the means bywhich the heart faces a hemodynamic burden, in addi-tion to the recruitment of neurohormonal mechanismsand the use of the Frank–Starling mechanism [1]. How-ever, load-induced hypertrophy (pathologic hypertrophy)is characterized by interstitial fibrosis, molecular remod-eling with reduced sarcoplasmic reticulum and increaseof sarcolemmal content of Ca2+ regulating proteins,and progressive cell death [2]. Several studies have doc-umented that post-ischemic recovery of mechanicalfunction is impaired in load-induced hypertrophy[3–6]. However, little data is available with regard tothe pathophysiological mechanisms of such impairedrecovery, particularly its dependency on perturbationsof intracellular Ca2+ handling and/or energy metabo-lism, nor myocardial TNF-a expression has been testedin response to acute mechanical stretch in hypertrophiedhearts.

On the other hand, myocardial growth stimulatedby the activation of the growth hormone/insulin-likegrowth factor I (GH/IGF-I) axis appears more ‘‘phys-iologic’’ than load-induced hypertrophy, at least in theshort-term, insofar as it is associated with unchangedcapillary density, no interstitial remodeling, normalor even augmented systolic and diastolic function,and reduced apoptotic rate [7,8]. Such observationshave prompted several experimental investigationsfocused on GH/IGF-I activation in heart failure bygenetic manipulation or pharmacological means[8–11].

However, no study has compared vis-a-vis the molec-ular and histological characteristics of GH- and load-induced hypertrophy. In addition, there is no informationas to whether these two kinds of hypertrophy entail dif-ferent responses to ischemic and mechanical injury. Thecurrent study was performed to clarify these issues usingclassical models of ischemia-reperfusion and mechanicalstretch in vitro.

2. Methods

The investigation conforms with the Guide for the

Care and Use of Laboratory Animals published by theUS National Institutes of Health (NIH PublicationNo. 85-23, revised 1996), and experimental procedureswere approved by the local Animal Care Committee.Wistar rats of male sex were used and the study designis shown in Fig. 1. Transverse banding of ascending

aorta (AB) was performed placing a tantalum hemoclipin rats aged 3 weeks, as previously described [12]. Shamrats underwent the same surgical procedures, althoughthe clip was not placed. Growth hormone treatment(3.5 mg/kg body weight rhGH per day via two subcuta-neous injections) or placebo (normal saline) were ran-domly started in rats aged 9 weeks and continued for4 weeks. Ex vivo studies were performed at 13 weeksof age. No death was observed during surgery and be-fore the final experiments. Timing of AB and GH treat-ment, and even more GH dosage, were established onthe basis of pilot experiments (unpublished data) show-ing that at 13 weeks of age, AB and GH-treated ratsachieved similar D left ventricular growth responses(see below).

2.1. Assessment of cardiac growth in AB and GH-treated

rats

Although the optimal method for comparing heartweights in the rat is unknown, normalizing left ventric-ular weight to tibial length relates cardiac size to theamount of lean body tissue and to cell size more thandoes normalization to body weight [13]. Accordingly,at the end of the final experiments, right hind legs ofthe rats were removed by disarticulating the femursfrom the acetabulum at the hip. The tibias were dis-sected free of soft tissue and frozen at �20 �C. Fourradiographic films (X-Omat XTL2, Eastman KodakCo) of the tibias were then obtained, and the tibiallength of each animal was assessed with a caliper fromthe radiograph.

2.2. Protocol A

A total of 32 hearts (n = 8 in each group) underwentbasal molecular analysis and histochemistry. Immedi-ately after rats were sacrificed, left ventricles were care-fully separated form right ventricles and stored forsubsequent analysis.

2.2.1. Basal myocardial mRNA expression of Ca2+

regulating proteins (n = 4 in each group)

Total RNA was prepared from left ventricular myo-cardium according to the method of Chomczynskiet al., as previously described [14]. For sarcoplasmicreticulum calcium-ATPase (SERCA-2), Na–Ca2+ ex-changer (NCX) and phospholamban (PLB), the poly-merase chain reaction products which were 523, 429and 535 bp respectively, were cloned into a Bluescriptvector. They were subsequently transformed into com-

Fig. 1. Study design. AB = aortic banding; GH = growth hormone.

H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40 31

petent bacteria which where selected and sequencedusing the Big Dye TM terminator cycle sequencing kit(ABI Prism, PE Applied Biosystems, Warrington,UK). RNAse protection assay (RPA II kit Ambion)and the solution hybridization RNAse protection assaywere performed according to the manufacturer�s instruc-tions with 20 lg of total RNA, prepared as previouslydescribed [14].

2.2.2. Collagen volume fraction (n = 4 in each group)

The whole left ventricles were cut serially into 10–14transverse sections (varying according to the heart�s size)1 mm thick each, from apex to base. Six lm thick trans-verse sections were subsequently stained with Picrosiriusred. Slides were observed with a Nikon Microphot FXAlight microscope equipped with a polarized set and ana-lysed with Zeiss KS300 software. Collagen volume frac-tion (CVF) was expressed as the mean percentage ofconnective tissue areas divided by total tissue area inthe same field [15].

2.3. Isolated whole heart preparation

A total of 64 rats (n = 16 in each group), underwentisolated whole heart preparation protocol (Fig. 1, Pro-tocol B and C). Under deep anesthesia, hearts were rap-idly excised and immersed in ice-cold Krebs–Henseleitbuffer, weighted and mounted in a Langendorff appara-tus at a constant temperature of 37 �C, as previouslydescribed [16–19]. Perfusion was set at a constant flow

of 12 ml/min per gram of heart weight by means of aroller pump using phosphate-free, Krebs–Henseleit buf-fer (118 mmol/l NaCl, 4.7 mmol/l KCl, 1.75 mmol/lCaCl2, 1.2 mmol/l MgSO4, 0.5 mmol/l EDTA,25 mmol/l NaHCO3, 11 mmol/l glucose), which wasinline filtered (45 lm pore size) and bubbled with 95%O2 and 5% CO2 to yield a pH of 7.4; coronary perfu-sion pressure was measured with a Statham P23Dbtransducer (Gould Instruments, Oxnard, CA, USA)connected to the perfusion line. To calculate the coro-nary flow, the total effluent was collected intermittentlyin a calibrated cylinder over a time period of 1 min.Cardiac temperature was measured by a temperatureprobe inserted into the right ventricle and was kept con-stant within ±0.1 �C by regulating the temperature ofthe perfusate. Left ventricular mechanical performancewas continuously measured with a water-filled latex bal-loon inserted into the left ventricle through an incisionin the left atrial appendage, via the mitral valve, and se-cured by a ligature. The balloon was attached to a stiffplastic tube and connected to a Statham P23Db pres-sure transducer (Gould Instruments, Oxnard, CA,USA) for continuous measurement of isovolumic pres-sure. The hearts were paced at 5 Hz using monophasicsquare-wave pulses delivered from a Grass Stimulator(model S 88 S1U5) and two pericardial electrodes onthe free wall of the right ventricle. Left ventricular pres-sure and coronary perfusion pressure curves were digi-tized using a commercially available computer. In orderto achieve comparable loading conditions (i.e., balloonvolumes) in hearts of different sizes and geometry, the

32 H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40

left ventricular parameter of interest was acquired withthe intraventricular balloon inflated at 50% of the vol-ume that resulted in the maximum left ventriculardeveloped pressure (50% of Volmax), as previously vali-dated [18]. Left ventricular wall stress (r) and relativewall thickness (hr) were derived from left ventricularpressure measurements, intraventricular balloon vol-ume, and weight of left ventricle as previously described[18].

2.4. Protocol B

A total of 48 hearts (n = 12 in each group), under-went ischemia-reperfusion protocol with assessment ofintracellular Ca2+ handling and high-energy phosphatemetabolism. After an equilibration period of 15 min at50% of Volmax, the perfusion line was clamped (no-flowischemia) and hearts were subjected to global normo-thermic ischemia for 20 min followed by 30 min ofreperfusion. Half of the hearts in each group were stud-ied in the Ca2+-setup, the other half in the 31P-NMRsetup.

2.4.1. Aequorin loading and quantification of intracellular

Ca2+ (n = 6 in each group)

Intracellular Ca2+[Ca2+]i was measured in parallelexperiments by aequorin normalized by fractional lumi-nescence as described below [17,19]. After stabilizationfor 10 min at 25 �C, 3–5 ll of an aequorin-containingsolution (1 lg/ml) were injected with a glass micropipetteinto the interstitium of the inferoapical region of the leftventricle. The heart was positioned in an organ bath withthe aequorin-loaded area of the left ventricle directed to-wards the cathode of a photomultiplier (model 9635QA,Thorn-EMI, Gencom, Inc., Fairfeld, NJ, USA) and sub-merged in Krebs–Henseleit solution. Subsequently, thetemperature was increased to 37 �C within 10 min andkept constant. Aequorin light signals were recorded onthe 4-channel recorder (Graphtec, Seefeld 82229, Ger-many), digitized by a 12 bit analog-digital convertingboard at a sampling rate of 1 kHz (DAP 800/3, Micro-star, Bellevue, WA, USA) and stored on the hard driveof a computer (PC 133 MHz). Normalization of aequo-rin light signals and analysis of calcium-overload in thefirst minute of reperfusion was performed as previouslydescribed [17,19]. Specifically, the following parameterswere used: [Ca2+]isch = end-ischemic [Ca2+]i, peak = ini-tial peak of Ca2+ signal in the first minute of reperfusion,Oscillsys = maximum of the first 10 transients of Ca2+

oscillations in the first minute of reperfusion (absolutevalue), I(0�60) = time integral of aequorin light signalfrom the beginning until 60 s of reperfusion, normalizedby Lmax [19]. A representative tracing showing an origi-nal pressure and light signals and the first 30 s of reper-fusion is depicted in Fig. 2.

2.4.2. 31P-NMR Spectroscopy (n = 6 in each group)

High-energy phosphates were measured by 31P nu-clear magnetic resonance (31P-NMR) on a 7 T BrukerAM system, as previously described [16]. Data wereaveraged over 5 min time intervals with 4 K datapoints, 152 scans, TR = 1.93 s. Peak amplitudes werecorrected for partial saturation (8% for Pi, 12% forPCr) by comparing to fully relaxed spectra. The con-centration of the gamma ATP in control hearts wasset to 10.8 mM [16] and normalized to the heart weightto obtain the relation between spectrometer intensitiesand absolute concentrations [20]. Concentrations of allother compounds were computed using this factor,taking into account the heart weight of each individualheart. Intracellular pH (pHi) was measured by compar-ing the chemical shift, d, between inorganic phosphate(Pi) and phosphocreatine (PCr) to values obtainedfrom a standard curve.

2.5. Protocol C

A total of 16 hearts (n = 4 in each group), under-went mechanical stretch protocol with assessment ofstretch-induced myocardial tumor necrosis-a (TNF-a)mRNA expression. After 15–30 min at 25 �C, the tem-perature was gradually increased to 37 �C and keptconstant. After 15 min stabilization period, the balloonwas further inflated to achieve 50% (control un-stretched myocardium) or 140% of Volmax (severestretch), corresponding in controls hearts to a diastolicpressure of 5–10 and 35–40 mmHg, respectively [21].This was done to compare hearts of different size atsimilar preload levels, insofar as it is well known thatload-induced hypertrophy is accompanied by elevatedleft ventricular chamber stiffness, and therefore choos-ing the same value of left ventricular diastolic pressurein controls and AB would have resulted in a signifi-cant understretching of AB hearts [18]. At 10 minintervals during experiments, a sample of the coronaryvenous effluent was collected in a calibrated cylinderover a period of 1 min for measuring the lactate pro-duction (Lactate Reagent; Sigma Chemical Co.). After40 min, respectively, the balloon was rapidly deflatedto a volume just enough to obtain a pressure signal.After 5 min stabilization, perfusion was terminatedand left ventricles, carefully separated from right ven-tricles, were quickly frozen in liquid nitrogen. TotalRNA was extracted from homogenized left ventricularmyocardium and Northern analysis was performed aspreviously described in details [21].

2.6. Statistical analysis

All data are presented as mean ± SEM. For statisti-cal comparison among the various groups of hearts

Fig. 2. Original tracing (upper panel) from a control heart showing coronary perfusion pressure (CPP), left ventricular pressure (LVP) and aequorinlight signal normalized to Lmax (L/Lmax), where control is representative of sham and placebo rats. The lower panel depicts the first 30 s ofreperfusion at an enlarged time scale. Note that reperfusion peaks consists of a sequence of Ca2+ transients, a phenomenon known as ‘‘Ca2+-oscillations’’.

H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40 33

the multiple comparison factorial-one factor ANOVAwas used. Significant differences were detected usingthe Neuman–Keuls test. A p values of <0.05 were con-sidered significant.

3. Results

3.1. Characteristics of cardiac growth

AB and GH rats had a similar left ventricular concen-tric hypertrophy, as shown by a comparable values ofboth left ventricular to tibial length ratio and thicknessto radius ratio, indicating similar myocardial growth re-sponses in AB and GH groups (Table 1). Molecularanalysis of left ventricular myocardial mRNA expres-sion of SERCA-2, PLB and NCX revealed significant

changes in the AB group, consistent with pathologic car-diac remodeling, while in the GH group message levelsof the above calcium regulating proteins were unaffected(Table 1). In addition, left ventricular myocardial CVFwas significantly increased in AB, while in the GH-treated animals it was unchanged (Fig. 3). Myocyte areawas slightly but not significantly increased in AB andGH rats (not shown).

3.2. Mechanical performance, calcium handling andhigh-energy phosphate metabolism

Data on baseline left ventricular mechanical perfor-mance are shown in Table 2. Developed pressure wassignificantly increased in the hypertrophied comparedwith control hearts. However, developed wall stress,an index which normalizes pressure to left ventricular

Tab

le1

Somatic

growth,morphometrichistology

andmyo

cardialmRNA

expressionofCa2

+regu

latinggenes

BW

(g)

TL(m

m)

LVW

(mg)

LVW/B

W(m

g/g)

LVW/T

L(m

g/mm)

hr

SERCA-2

(pg/lg

RNA)

PLB(pg/lgRNA)

NCX

(pg/lgRNA)

CVF

(%)

N48

4848

4848

328

88

8Sham

376±

1839

.4±

0.5

979±

562.51

±0.08

24.3

±0.9

0.99

±0.05

4.31

±0.05

1.38

±0.11

0.36

±0.03

3.8±

0.1

AB

361±

1438

.9±

0.3

1336

±94

*3.70

±0.11

*34

.3±

1.8*

1.23

±0.03

*3.21

±0.17

*0.43

±0.17

*0.51

±0.02

*7.7±

0.3*

N48

4848

4848

328

88

8Placebo

383±

1338

.8±

0.4

967±

692.60

±0.05

24.9

±1.4

1.07

±0.03

4.37

±0.02

1.31

±0.17

0.39

±0.01

3.6±

0.3

GH

441±

16*,**

40.8

±0.5*

,**

1298

±78

*2.94

±0.07

*,**

32.3

±1.5*

1.18

±0.02

*4.26

±0.05

**

1.25

±0.18

**

0.38

±0.03

**

3.7±

0.1*

*

Dataaremean±

SEM;N

=number

ofan

imal

considered

foreach

variab

le;AB=

aorticban

ding;

GH

=growth

horm

one;BW

=bodyweigh

t;TL=

tibiallength;LVW

=leftventricularweigh

t,expressed

asan

atomical

wet

weigh

t;hr=

relative

wallthickness;

SERCA-2

=sarcoplasm

icreticulum

calcium-A

TPase;

PLB=

phospholamban

;NCX

=Na+

–Ca2

+exchan

ger;

CVF=

collag

ene

volumefraction.

*p<0.05

vs.sham

andplacebo.

**p<0.05

vs.AB.

34 H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40

weight and geometry, was not significantly differentamong the three groups, indicating similar left ventricu-lar intrinsic contractility. While in GH treated rats therewas no evidence of diastolic impairment, in pressure-overloaded animals end-diastolic pressure, time to 90%relaxation, and Tau at 50% of Vmax were significantlyhigher compared with controls. The perfusion pressurein the GH and control group was 80 ± 2 (mean ± SE)and 81 ± 4 mmHg, respectively, while in AB group itwas 98 ± 5 mmHg. During baseline perfusion, theCa2+ transients were not significantly different amongthe three groups; however, there was a tendency to aprolonged Ca2+ transient in the AB group, which didnot come out statistically significant (Fig. 4). After theonset of global ischemia, hearts stopped beating within2 min. During ischemia, contracture started afterapproximately 10 min in all groups and end-diastolicpressure showed the well-described overshoot uponreperfusion (Fig. 5). Recovery of developed and end-diastolic pressures were both markedly altered in theAB group, compared with GH and control hearts(Fig. 5). Heart rate was similar in all groups duringthe ischemia protocol.

Quantitative high energy phosphate data from thewhole experimental protocol are depicted in Table 2and Fig. 5. Under baseline perfusion, PCr content waslower in the AB group and Pi was increased comparedwith the control group, whereas ATP was similar. GHtreated rats exhibited no differences regarding ATP,PCr or Pi as compared with controls, but had lowerPCr and Pi content than AB rats. No differences inintracellular pH were evident during baseline perfusionamong the three study groups. PCr fell rapidly below20% of its baseline value within 5 min and was undetect-able after 15 min of ischemia. Pi started to increase sev-eral fold to values as high as 25 mM at the end ofischemia. ATP decreased to about 2 mM after 15 minof ischemia, however, the time course was slower thanthat of the PCr decrease. The time courses of metabolicchanges did not differ during ischemia among the threestudy groups. Intracellular pH fell to 6.0 at the end ofischemia and recovered during reperfusion within5 min to control values in all groups. Post-ischemicrecovery of PCr as percentage of the pre-ischemic valuewas similar (about 70%) in all groups, although absolutePCr content was reduced in the AB group (Fig. 5). ATPrecovered to the same extent in control, AB, and GHgroups; (40 ± 5, 39 ± 3, and 34 ± 6%, respectively)and Pi decreased, but remained elevated to about350% of pre-ischemic values. Maximal intracellularCa2+ overload occurring in the first minute of reperfu-sion as defined by Oscillsys showed a 1.6-fold increasein the AB hearts (Fig. 6). [Ca2+] at the end of ischemia,peak [Ca2+] or integrated light emission during the firstminute of reperfusion showed no differences betweengroups.

Fig. 3. Left ventricular collagen deposition stained with picrosirius red in control, aortic banding (AB) and growth hormone (GH) group, wherecontrol is representative of sham and placebo rats. Note the marked increase of the collagen framework surrounding cardiomyocytes in the groupsubjected to AB.

Table 2Left ventricular mechanical performance and high energy phosphate metabolism during baseline perfusion

dP (mmHg) dr (kdynes/cm2) ed-P (mmHg) T90R (ms) Tau (ms) ATP (mmol/L) PCr (mmol/L) Pi (mmol/L) pHi

N 12 12 12 12 12 12 12 12 12Sham 86 ± 5 29 ± 3 8 ± 1 77 ± 5 39 ± 5 10.6 ± 0.2 14.4 ± 0.6 2.7 ± 0.3 7.1 ± 0.01AB 117 ± 2* 28 ± 2 13 ± 2* 94 ± 4* 52 ± 2* 10.3 ± 0.6 11.9 ± 0.9* 4.8 ± 0.7* 7.1 ± 0.01

N 12 12 12 12 12 12 12 12 12Placebo 89 ± 2 27 ± 1 6 ± 2 72 ± 3 41 ± 2 10.1 ± 0.7 14.9 ± 0.2 2.4 ± 0.5 7.1 ± 0.02GH 110 ± 3* 31 ± 1 6 ± 1** 71 ± 3** 39 ± 3** 11.1 ± 0.5 15.3 ± 0.6** 2.6 ± 0.4** 7.1 ± 0.01

Data are mean ± SEM; N = number of animal considered for each variable; AB = aortic banding; GH = growth hormone; dP = developed pres-sure; dr = developed wall stress; ed-P = end-diastolic pressure; T90R = time to 90% relaxation; PCr = phosphocreatine; Pi = inorganic phosphate.* p < 0.05 vs. sham and placebo.

** p < 0.05 vs. AB.

H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40 35

3.3. Stretching protocol

Under basal conditions, no myocardial TNF-amRNA expression was found in control and GH leftventricles, whereas detectable TNF-a message levelswere noted in myocardium from pressure-overloadedanimals (Fig. 7). No change in basal myocardial TNF-a mRNA expression was noted during perfusion withmaintenance of the uninflated balloon (�5 mmHg leftventricular end-diastolic pressure). On the contrary,myocardial stretch at 140% of Vmax, corresponding toa diastolic pressure of 40 mmHg in controls and GHanimals, and to 63 mmHg in the AB group, was associ-ated with de novo myocardial TNF-a expression in bothcontrols and GH hearts, with a further and remarkableincrease in TNF-a transcript levels in hypertrophiedhearts subjected to AB (�5-fold vs. baseline; p < 0.001,Fig. 7). No lactate production was found in the coro-nary venous effluent during or after 40 min of stretch(data not shown).

4. Discussion

The main findings of the current study are: (i) patho-logic left ventricular hypertrophy induced by pressureoverload is characterized by increased collagen volumefraction, diastolic dysfunction, significant changes in

the myocardial mRNA expression of key Ca2+ regulat-ing proteins, and greater vulnerability to ischemia-reperfusion injury and mechanical stretch comparedwith normal hearts; (ii) by contrast, GH-induced myo-cardial growth appears to be substantially different innature insofar as is not associated with fibrosis andchanges in Ca2+ regulating proteins, and diastolic func-tion is preserved. Importantly, the susceptibility toischemia-reperfusion and mechanical stretch in vitro isnot significantly altered by GH-induced myocardialgrowth. The data support the concept that this kind ofgrowth is not detrimental but rather confers cardiopro-tection against ischemic and mechanical injury com-pared with pressure-overload hypertrophy.

4.1. Current study

The current study provides a potential explanationfor the altered post-ischemic recovery of left ventricularfunction in load induced hypertrophy. Indeed, directmeasurement of intracellular Ca2+ showed a marked in-crease of Ca2+ overload in AB hearts upon reperfusion.Oscillsys reflects the amount of Ca2+ released by theoverloaded sarcoplasmic reticulum on top of the cyto-solic Ca2+-overload, and are likely generated by rapidCa2+ release and re-uptake by the sarcoplasmicreticulum since inhibitors of the sarcoplasmic reticulumabolishes them [19]. Ca2+ oscillations in turn are known

Fig. 4. Representative signal averaged Ca2+ transients (upper panel)and corresponding mechanical contractions (lower panel) of sham,growth hormone treated (GH) and hearts with hypertrophy due toaortic banding (AB), where sham is also representative of placebo rats.Pressure development is similarly increased in both hypertrophygroups compared to sham. Diastolic function is impaired in AB butnot in GH rats. The Ca2+ transients were not significantly different,however there was a tendency of a Ca2+ transient prolongation in theAB group.

36 H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40

to induce reperfusion arrhythmias, particularly ventricu-lar fibrillation and myocardial dysfunction.

Impaired myocardial high energy phosphate metabo-lism was also explored as an alternative potential culpritof the increased post-ischemic left ventricular dysfunc-tion. However, this mechanism appears unlikely, giventhe similar time-course and percent recovery of PCr,ATP, and Pi content among the three study groups,despite baseline differences. The negative impact of

load-induced hypertrophy on the myocardium is furthersupported by the stretching experiments. MyocardialTNF-a mRNA was already detectable under baselineconditions in the AB group, at variance with GH-trea-ted and control animals. Moreover, its upregulation inresponse to mechanical stretch was much more markedthan that occurring in the other study groups. This find-ing bears relevant implications in view of the importantrole that TNF-a appears to play in the pathogenesis ofheart failure. Indeed TNF-a stimulates myocyte apopto-sis, decreases contractility, and disrupts the extracellularmatrix via the activation of metalloproteases [22,23].

Contrary to the situation with pressure overloadhypertrophy, the current study supports the notion thatGH-induced myocardial growth displays unique physio-logic features. Indeed, not only was the extracellular ma-trix integrity maintained in GH treated rats and nochanges were observed in candidate Ca2+ regulatinggenes, but also the susceptibility to ischemia-reperfusioninjury and mechanical stretch was similar to normalhearts.

4.2. Comparison with previous studies

Left ventricular hypertrophy induced by chronichemodynamic overload is commonly accompanied byfibrosis, decreased capillary density, and decreased en-ergy reserve for use under stress situations [2,20]. Inaddition, hypertrophic hearts exhibit an impaired func-tional recovery from ischemia-reperfusion injury [3–6].Allard et al. have suggested the possibility that Ca2+

overload may be augmented during reperfusion,although no direct measure of intracellular Ca2+ wasprovided in that study [3]. As to myocardial metabolism,our findings of decreased PCr during baseline perfusionare in agreement with those reported by Tian et al., whofound the energy reserve to be decreased during baselineperfusion in hearts with pressure overload hypertrophy[20]. No data concerning high energy phosphate metab-olism after global ischemia was reported in that study.On the contrary, despite significant hypertrophy, GHtreated hearts displayed indices of high energy phos-phate metabolism not different from control hearts.Such favorable energetic profile was described originallyby Mercadier�s group, who proposed an increased num-ber of active cross-bridges despite a reduced cross-bridgecycling velocity, typical of V3 isomyosin, in hearts sub-jected to chronic GH hypersecretion [24].

Early upregulation of the TNF-a activity has beenconsistently documented within the stressedmyocardium after persistent mechanical stimulation inend-stage human heart failure and after experimentalload-induced hypertrophy, and in myocardial infarction[22,23,25]. In particular, myocardial generation of TNF-a following mechanical stress was recently characterizedby our group in the same model system, showing that it

Fig. 5. Mechanical function and high energy phosphate metabolism in the sham, aortic banding (AB) and growth hormone (GH) group, where shamis also representative of placebo rats. Note the decreased post-ischemic recovery of left ventricular function and the higher end-diastolic pressure inthe aortic banding (AB) group (upper panels). PCr content was decreased at baseline and reperfusion in the banded animals, but percent recovery issimilar in all groups (lower left panel). ATP baseline differences were not evident upon reperfusion (lower right panel). * p < 0.05 vs. sham; � p < 0.05vs. GH.

H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40 37

is not constitutively expressed in the myocardium undernormal circumstances [21]. Conversely, graded mechan-ical stretch resulted in a progressive increase in TNF-amRNA and protein levels. No data were available asto TNF-a in an already hypertrophic myocardium.Interestingly, the fact that myocardial TNF-a mRNAexpression was remarkably upregulated in response tosevere mechanical stretch suggests that load-inducedmyocardial hypertrophy has an increased susceptibilityto hemodynamic challenges, which may promote andsustain the transition towards the heart failurephenotype.

In the short-term, GH-induced left ventricularhypertrophy is characterized by unchanged capillarydensity, absence of interstitial fibrosis, and normal dia-stolic function, a picture supported by both animal

and human studies [7,26,27]. By contrast, in thelong-term GH hypersecretion is associated with thetypical stigmata of acromegalic cardiomyopathy, i.e.marked left ventricular enlargement, impaired systolicand diastolic function, and interstitial fibrosis [28]. Ta-ken together, our findings lend further support to thehypothesis that, in the short-term, GH/IGF-1 axisactivation induces a unique type of myocardial re-sponse that shares many features with physiologicalgrowth.

Our data are in agreement and expand on those re-ported by Ross Jr.�s group, showing that exogenousGH/IGF-1 in mice produce cardiac hypertrophy and apositive inotropic effect without causing significantchanges in expression of fetal and other selected myo-cardial genes [29].

Fig. 6. Indices of Ca2+ overload (see Section 2 for details) in the firstminute of reperfusion in control, aortic banding (AB) and growthhormone (GH) group, where control is representative of sham andplacebo rats. The first 10 systolic Ca2+ oscillations were increased 1.6-fold in the aortic banding (AB) group vs. controls. * p < 0.05 vs.controls; � p < 0.05 vs. GH.

Fig. 7. Representative Northern blot and densitometric analysis ofautoradiographic bands, expressed as mean ± SEM, for TNF-aexpression in unstretched (50% of Volmax) and severely stretched(140% of Volmax) myocardium from controls (n = 8), growth hormone(GH, n = 4) and aortic banding (AB, n = 4) rats, where control isrepresentative of sham and placebo rats. Twenty-five micrograms oftotal RNA was loaded for each lane. Probe control lane refers toendotoxin-stimulated (10 lg/mL for 8 h) rat macrophage cells. Fold-induction was referred to an arbitrary number, defined as 1, assignedto the level of expression estimated in severely stretched myocardiumfrom control rats. Note the mechanical stretch-induced de novo

myocardial TNF-a mRNA expression in GH group and controls,which was dramatically increased in AB animals. * p < 0.001 vs. thecorresponding stretched groups; � p < 0.001 vs. the correspondingunstretched myocardium.

38 H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40

4.3. Pathophysiological and clinical implications

The current study provides two mechanisms poten-tially responsible for the poor outcome of pathologic leftventricular hypertrophy, namely intracellular Ca2+

overload during ischemia and enhanced stretch-inducedmyocardial TNF-a expression. Not only is thisparticular type of myocardial growth characterized byincreased fibrosis, reduced capillary density, re-expres-sion of the embryonic gene program, but it is also par-ticularly susceptible to frequently occurring challenges.In the clinical scenario, repetitive episodes of ischemiamay often occur in hypertrophied hearts, as well as sud-den left ventricular mechanical stretch due to hyperten-sive bursts, or a combination thereof. Such events, byincreasing intracellular Ca2+ overload and TNF-a myo-cardial production, may induce lethal ventriculararrhythmias and accelerate the transition to heartfailure.

It is controversial whether reactivation of cardiacgrowth may be beneficial in heart failure. Recent studiesusing molecular techniques have shown that inhibitingmyocardial growth by genetic manipulation, such asblocking Gaq or RAS signaling prevents cardiac dys-function despite increased wall stress [30,31]. Con-versely, stimulation of cardiac growth, particularlywhen oriented toward concentric remodeling, by activa-tion of ERK, telomerase reverse transcriptase, or inhibi-tion of nitric oxide synthesis appears to protect fromcardiac decompensation in similar models of pressureoverload [32]. The current data point to the existenceof peculiar types of ‘‘physiologic’’ myocardial growththat may be beneficial in the setting of cardiac remodel-ing given the striking differences with pressure overloadhypertrophy. In this regard, GH-induced myocardial

growth also stimulates survival pathways, as recentlysuggested by two studies in the rat model of post-infarc-tion heart failure [15,33]. The complex intracellular sig-naling involved in these such salutary actions is underactive investigations and possibly involves Akt, a specificsubstrate of IGF-I positioned downstream of PI3-kinase[34]. In fact, relevant to the current study, Akt-inducedhypertrophy is associated with a positive inotropic effectwithout re-expression of the fetal gene program andp44/42 MAPK activation in vivo, at variance withstress-induced pathologic hypertrophy [34]. In addition,more recently, McMullen et al. have shown that trans-genic mice overexpressing IGF-1 receptor in the heartdisplayed compensated cardiac hypertrophy, character-ized by increased myocyte size with no evidence of histo-pathology and enhanced systolic function over time,

H. Stromer et al. / Growth Hormone & IGF Research 16 (2006) 29–40 39

which was associated with a significant activation of thePI3-kinase/Akt-p70S6K1 pathway [35].

Acknowledgements

The MRUI software package was kindly provided byA. van den Boogaart, Katholieke Universiteit Leuven. Itis currently funded by the EC project TMR/NetworksERB-FMRX CT970160 (http://azur.univ-lyon1.fr/TMR/tmr.html). Human recombinant growth hormonewas kindly supplied by Pharmacia & Upjohn GmbH,Erlangen Germany.

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